RT Journal Article
SR Electronic
T1 Evidence for a Replication Function of Ffa-1, the Xenopus Orthologue of Werner Syndrome Protein
JF The Journal of Cell Biology
JO J Cell Biol
FD Rockefeller University Press
SP 985
OP 996
DO 10.1083/jcb.152.5.985
VO 152
IS 5
A1 Chen, Chin-Yi
A1 Graham, Jeanine
A1 Yan, Hong
YR 2001
UL http://jcb.rupress.org/content/152/5/985.abstract
AB DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form “hybrid foci,” which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.